The fungus Candida albicans causes diverse infections with substantial morbidity and mortality. The organism remains a threat for many reasons, including the limited spectrum of antifungal drugs, the occurrence of drug resistance, and the array of virulence determinants that enable it to infect almost any tissue. Much of our mechanistic understanding of C. albicans infection biology comes from analysis of one clinical isolate, strain SC5314, and its derivatives. One chronic knowledge gap in the field is the extent to which conclusions from analysis of SC5314 can be generalized to other clinical isolates. For C. albicans, as for most pathogens, there is considerable phenotypic diversity among clinical isolates. On that basis, we believe that it is critical to validate gene functions with a panel of C. albicans strain isolates in order to prioritize genes and pathways for further study and therapeutic targeting. Our goal is to develop a toolbox that enables rapid and precise genetic manipulation of diverse clinical C. albicans isolates. We will use the recently described C. albicans CRISPR-Cas9 system along with a marker excision system we have just developed. We seek to create a system for rapid and rigorous genetic assays in clinical C. albicans isolates through implementation first in the cornerstone clinical isolate, SC5314, where we will make an appraisal consistency and off-target effects of our procedure. We will then move to a panel of clinical isolates as a test case for appraisal of natural variation in gene function, using the well-studied virulence regulator Efg1 for a test case. Proposed studies will provide a paradigm for multi-strain gene function validation in C. albicans, and will yield materials and methods to enable implementation in the community. In addition, our marker recycling approach relies upon conserved cellular machinery, and thus may be useful in many other organisms.